WO2018224951A2 - Anticorps multispécifiques modifiés et autres protéines multimères avec des mutations de région ch2-ch3 asymétriques - Google Patents

Anticorps multispécifiques modifiés et autres protéines multimères avec des mutations de région ch2-ch3 asymétriques Download PDF

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WO2018224951A2
WO2018224951A2 PCT/IB2018/053997 IB2018053997W WO2018224951A2 WO 2018224951 A2 WO2018224951 A2 WO 2018224951A2 IB 2018053997 W IB2018053997 W IB 2018053997W WO 2018224951 A2 WO2018224951 A2 WO 2018224951A2
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nos
region
protein
antibody
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WO2018224951A3 (fr
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Mark Chiu
Adam ZWOLAK
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Janssen Biotech, Inc.
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Priority to AU2018281045A priority Critical patent/AU2018281045A1/en
Priority to KR1020197038922A priority patent/KR20200014379A/ko
Priority to JP2019566965A priority patent/JP2020522266A/ja
Priority to BR112019025583-4A priority patent/BR112019025583A2/pt
Priority to CN201880037442.XA priority patent/CN110785185A/zh
Priority to EP18814314.3A priority patent/EP3634486A4/fr
Priority to CA3065171A priority patent/CA3065171A1/fr
Priority to RU2019144115A priority patent/RU2804031C2/ru
Priority to MX2019014576A priority patent/MX2019014576A/es
Publication of WO2018224951A2 publication Critical patent/WO2018224951A2/fr
Publication of WO2018224951A3 publication Critical patent/WO2018224951A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to engineered multispecific antibodies and other multimeric proteins with asymmetrical CH2-CH3 region mutations and methods of making and using them. SEQUENCE LISTING
  • bispecific antibodies for dual- targeting, cell redirection efforts, and immune checkpoint modulation; indeed many bispecific therapeutics are currently in clinical trials (Jachimowicz et al. BioDrugs. 2014 (4):331-43).
  • the development of bispecific antibodies has been limited by the difficulty of both upstream and downstream processes, being able to generate high titers and pure product in a reproducible and scalable manner, and separating bispecific molecules from excess parental or intermediate molecules.
  • Methods for specifically pairing IgG heavy chains or half molecules have been developed, and include knob-in-holes, controlled Fab arm exchange, CrossMAb, and common light chains and orthogonal Fab interface.
  • Production of Fv-based molecules i.e. BiTEs, Diabodies
  • non-IgG based scaffolds i.e. DARPins, Adnectins, fynomers, and centyrins
  • Fv-based molecules i.e. BiTEs, Diabodies
  • non-IgG based scaffolds
  • Fv-only or alternative scaffold-based molecules are their typically shorter serum lifetimes resulting from urinary excretion or from lysosomal degradation due to their inability to be recycled by FcRn.
  • IgG-based multispecific molecules containing an intact Fc domain are attractive based on their longer serum half-lives, ability to facilitate effector functions, and induction of apoptotic pathways.
  • bispecific antibodies can be challenging due to the multiple steps required to remove residual parental and other intermediate mAbs and Ab fragment molecules. Such molecules can have biophysical characteristics that are similar to the derived bispecific antibodies and thus cannot be easily separated by chromatographic methods. This difficulty in purification can lead to either a decrease in yield or purity of the bispecific molecule.
  • the invention provides for an isolated multispecific antibody comprising a first CH2- CH3 region comprising a mutation Q311R, Q31 IK, T307P/L309Q, T307P/V309Q,
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation Q31 IK and a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first
  • CH2-CH3 region comprising a mutation T307P/L309Q and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation T307P/V309Q and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation T307P/L309Q/Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation T307P/V309Q/Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated polynucleotide comprising the polynucleotide encoding the first CH2-CH3 region comprising a mutation Q31 IR.
  • T307P/V309Q/Q31 IR and the second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311; or
  • the invention also provides for a vector comprising
  • the isolated polynucleotide encoding the first CH2-CH3 region comprising a mutation Q31 IR, Q31 IK, T307P/L309Q, T307P/V309Q, T307P/L309Q/Q31 IR or
  • the isolated polynucleotide comprising a polynucleotide sequence of SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 87, 88 or 91;
  • the isolated polynucleotide comprising the polynucleotide encoding the first CH2-CH3 region comprising a mutation Q31 IR, Q31 IK, T307P/L309Q, T307P/V309Q.
  • the invention also provides for a host cell comprising the vector of the invention.
  • the invention also provides for a method of making the isolated multispecific antibody of the invention, comprising
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain comprising a mutation Q31 IR, Q31 IK, T307P/L309Q,
  • T307P/V309Q T307P L309Q/Q31 IR or T307P/V309Q/Q31 IR and a second heavy chain comprising wild-type amino acid residue at positions 307, 309 and 311, comprising
  • a second parental antibody comprising the second heavy chain comprising wild-type amino acid residue at positions 307, 309 and 311 and a second light chain; contacting the first parental antibody and the second parental antibody in a sample; incubating the sample; and
  • the invention also provides for an isolated antibody comprising two heavy chains or fragments thereof having identical amino acid sequences and two light chains or fragments thereof, wherein the two heavy chains comprise a mutation Q31 IR, Q31 IK, T307P/L309Q, T307P/V309Q, T307P L309Q/Q31 IR or T307P/V309Q/Q31 IR, wherein residue numbering is according to the EU Index.
  • the invention also provides for a polynucleotide encoding the antibody heavy chain comprising the CH2-CH3 region of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 52, 53 or 56; or
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation Q311R, Q31 IK, T307P L309Q, T307P/V309Q, T307P L309Q/Q311R or
  • T307P/V309Q/Q311R and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for pharmaceutical composition comprising the multimeric protein of the invention.
  • the invention also provides for a method of making an isolated multimeric protein comprising a first CH2-CH3 region comprising a mutation Q311R, Q31 IK, T307P/L309Q,
  • T307P/V309Q T307P L309Q/Q311R or T307P/V309Q/Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, comprising
  • a first parental protein comprising the first CH2-CH3 region comprising the mutation Q311R, Q311K, T307P L309Q, T307P/V309Q, T307P L309Q/Q311R or T307P/V309Q/Q311R;
  • Figure 1A shows the alignment between human IgGl and mouse IgG2a CH2 domains from amino acid residues 305 to 315; residue numbering according to the EU Index.
  • Figure IB shows the interactions of IgGl CH2 residues T307, L309, and Q311 (underlined residues in the Figure) with FcRn or Z-domain (Z34C peptide). Each residue made side-chain interactions with residues in FcRn and with Z-domain. T307 interacted with II on the ⁇ 2 microglobulin domain of FcRn. L309 and Q311R were responsible for interactions with both FcRn and Z-domain (dashed and solid lines for L309 and Q311, respectively).
  • IQRT SEQ ID NO: 102 (portion of ⁇ 2 chain of FcRn); LNGEEFMDFDLKQGTWGGDWPEA: SEQ ID NO: 103 (portion of a chain of FcRn); VLTVLHQDWLN: SEQ ID NO: 104 (portion of IgGl CH2 domain); FNMQCQRRFYE ALHDPNLNEEQRNAKIKSIRDD C : SEQ ID NO: 99.
  • Figure 2A shows a dose response curve of competition binding of indicated monospecific antibodies with the mAb RSV-L for FcRn using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 2B shows a dose response curve of competition binding of indicated monospecific or bispecific antibodies with the mAb RSV-L for FcRn using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 3A shows a dose response curve of competition binding of indicated monospecific or bispecific antibodies with the mAb RSV-L for FcyRI using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 3B shows a dose response curve of competition binding of indicated monospecific or bispecific antibodies with the mAb RSV-L for FcyRIIa using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 3C shows a dose response curve of competition binding of indicated monospecific or bispecific antibodies with the mAb RSV-L for FcyRIIb using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 3D shows a dose response curve of competition binding of indicated monospecific or bispecific antibodies with the mAb RSV-L for FcyRIIIa using AlphaScreen assay.
  • the graph displays % maximum signal plotted vs concentration of competitor.
  • Figure 4A shows hydrophobic interaction chromatography (HIC) chromatogram demonstrating that a bispecific antibody can be separated from parental monospecific mAbs under conditions developed.
  • HIC hydrophobic interaction chromatography
  • Figure 4B shows HIC chromatogram of the sample of mixture of equimolar amount of antibodies RSV-L[TLQ] and gpl20-R and bsRSV-L[TLQ] generated using Fab arm exchange.
  • Figure 4C shows the elution profile of a sample of a mixture of antibodies RSV-L[TLQ], gpl20- R and bsRSV-L[TLQ] generated using Fab arm exchange from protein A resin.
  • Figure 4D shows HIC chromatogram of protein A elution peaks.
  • Figure 5A shows the elution profile of a sample of in-supernatant Fab arm exchanged bsRSV- L[TLQ].
  • Figure 5B shows HIC analyses of protein A affinity column pH 4.7 eluates of a sample from in- supernatant Fab arm exchanged bsRSV-L[TLQ].
  • Figure 5C shows HIC analyses of protein A affinity column pH 4.2 eluates of a sample from in- supernatant Fab arm exchanged bsRSV-L[TLQ].
  • Figure 5D shows HIC analyses of protein A affinity column pH 3.4 eluates of a sample from in- supernatant Fab arm exchanged bsRSV-L[TLQ].
  • Figure 6A shows protein A chromatogram of a sample of in-supernatant Fab arm exchanged bsRSV-L[Q311R] showing three distinct peaks eluting at pH 4.6, 4.2 and 3.4.
  • Figure 6B shows HIC analyses of protein A affinity column pH 4.6 eluates of a sample from in- supernatant Fab arm exchanged bsRSV-L[Q311R].
  • Figure 6C shows HIC analyses of protein A affinity column pH 4.2 eluates of a sample from in- supernatant Fab arm exchanged bsRSV-L[Q311R].
  • Figure 6D shows HIC analyses of protein A affinity column pH 3.4 eluates of a sample from in- supernatant Fab arm exchanged bsRS V-L [Q311R] .
  • Figure 7A shows protein A chromatogram of a sample of bsTNF-[TLQ] generated using common light chain technology.
  • Figure 7B shows HIC analyses of protein A affinity column pH 4.7 eluates of a sample of bsTNF-[TLQ] generated using common light chain technology.
  • Figure 7C shows HIC analyses of protein A affinity column pH 4.2 eluates of a sample of bsTNF-[TLQ] generated using common light chain technology.
  • Figure 7D shows HIC analyses of protein A affinity column pH 3.4 eluates of a sample of bsTNF-[TLQ] generated using common light chain technology.
  • Figure 8 shows the pharmacokinetic analysis of indicated antibodies in Tg32 hemizygous mice.
  • the graph displays the concentration of each mAb normalized to the initial time point of the linear phase plotted vs time. Each point represents mean + standard error of four animals per group.
  • Multimeric protein refers to a protein that is composed of two or more separate polypeptide chains that combine to form a single protein.
  • the polypeptide chains may be coupled non-covalently or covalently for example via disulfide bonds.
  • Bind refers to specific binding of two proteins, such as binding of an antibody to an antigen or binding of a multispecific protein to its ligand.
  • Specific binding refers to preferential binding of the two proteins with typically an equilibrium dissociation constant (K D ) of about lxlO "8 M or less, for example about lxlO "9 M or less, about lxlO "10 M or less, about lxlO "11 M or less, or about lxlO "12 M or less, typically with the K D that is at least one hundredfold less than its K D for binding to a non-specific antigen (e.g., BSA, casein).
  • K D equilibrium dissociation constant
  • Reduced binding refers to a measurable reduction in binding of the antibodies or the multispecific proteins of the invention having at least one mutation in the CH2-CH3 region to protein A ligand when compared to the binding of the parental molecule without the mutation.
  • Modulates binding refers to a measurable difference in binding of the antibodies or the multispecific proteins of the invention having at least one mutation in the CH2-CH3 region to FcyR or FcRn.
  • Antigen refers to a molecule, such as protein or a fragment of a protein that is capable of mounting an immune response in a subject.
  • Asymmetric stabilizing mutations refers to mutations in a first CH2-CH3 region and in a second CH2-CH3 region which are at different positions in the first and in the second CH2- CH3 region and favor (e.g. stabilize) heterodimer formation between the first CH2-CH3 region and the second CH2-CH3 region over homodimer formation between the first CH2-CH3 region or the second CH2-CH3 region.
  • Heterologous protein refers to a polypeptide or protein that is not naturally part or portion of a polypeptide comprising a CH2-CH3 region in an endogenous cell.
  • Fib ronectin type III (FN3) domain refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175: 1910-1918, 1993; Watanabe et al, J Biol Chem 265: 15659-15665, 1990).
  • Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fib ronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419.
  • Individual FN3 domains are referred to by domain number and protein name, e.g., the 3 rd FN3 domain of tenascin (TN3), or the 10 FN3 domain of fibronectin (FN10).
  • FN3 domains can ben engineered to bind an antigen with high specificity and affinity.
  • “Fynomer” refers to an antigen-binding protein derived from human Fyn SH3 domain that can be engineered to bind an antigen with high specificity and affinity.
  • Antibodies is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, rabbit, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, monospecific, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.
  • Fully length antibodies are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (for example IgM).
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge CH2 and CH3).
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the VH and the VL may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR complementarity determining regions
  • ImMunoGeneTics database; Web resources, http://www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs.
  • IMGT ImMunoGeneTics
  • CDR CDR
  • HCDR1 CDR1
  • HCDR2 CDR3
  • LCDRl CDR 2
  • LCDR3 CDR3
  • Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant region amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa ( ) and lambda ( ⁇ ), based on the amino acid sequences of their constant regions.
  • Antigen-binding fragment refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody.
  • Exemplary antigen- binding fragments are heavy chain complementarity determining regions (HCDR) 1, 2 and/or 3, light chain complementarity determining regions (LCDR) 1, 2 and/or 3, the VH, the VL, the VH and the VL, Fab, F(ab')2, Fd and Fv fragments as well as domain antibodies (dAb) consisting of either one VH domain or one VL domain.
  • the VH and the VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs in which the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate chains, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Pat. Publ. No. WO1998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No. WO1994/13804; Int. Pat. Publ. No. WO1992/01047.
  • scFv single chain Fv
  • CH2-CH3 region refers to a portion of a human antibody constant domain and includes amino acid residues 231-446 (residue numbering according to the EU Index).
  • the CH2-CH3 region may have the C-terminal lysine at position 447 deleted.
  • “Monoclonal antibody” refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain.
  • Monoclonal antibodies typically specifically bind one antigenic epitope, except that bispecific or multispecific monoclonal antibodies specifically bind two or more distinct antigenic epitopes.
  • Monoclonal antibodies may have heterogeneous glycosylation within the antibody population.
  • Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.
  • Isolated refers to a homogenous population of molecules (such as synthetic polynucleotides or a protein such as an antibody) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step.
  • molecules such as synthetic polynucleotides or a protein such as an antibody
  • Isolated antibody refers to an antibody that is substantially free of other cellular material and/or chemicals and encompasses antibodies that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.
  • Humanized antibody refers to an antibody in which CDR sequences are derived from non-human species and the frameworks are derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or human immunoglobulin germline gene sequences.
  • Human antibody refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human germline immunoglobulin sequences. If the antibody contains a constant region or a portion of the constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • Human antibody comprises heavy or light chain variable regions that are "derived from” human germline immunoglobulin sequences if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin genes.
  • Such exemplary systems are human immunoglobulin gene libraries displayed on phage or mammalian cells, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci.
  • "Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to, for example introduction of somatic mutations, intentional introduction of substitutions into the framework or CDRs, and amino acid changes introduced during cloning and VJD recombination in non-human animals.
  • Human antibody is typically about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin sequences.
  • human antibody may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., JMol Biol 296: 57-86, 2000, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al, JMol Biol 397: 385-396, 2010 and in Int. Patent Publ. No.
  • Recombinant refers to antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means.
  • Multispecific refers to a protein, such as an antibody, that specifically binds two or more distinct antigens or two or more distinct epitopes within the same antigen. Multispecific protein may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis
  • Bispecific refers to a protein, such as an antibody, that specifically binds two distinct antigens or two distinct epitopes within the same antigen.
  • Bispecific protein may have cross- reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset), or may bind an epitope that is shared between two or more distinct antigens.
  • homologs such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset), or may bind an epitope that is shared between two or more distinct antigens.
  • “Monospecific” refers to a protein, such as an antibody, that specifically binds one distinct antigen or a distinct epitope.
  • Monospecific protein may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset), or may bind an epitope that is shared between two or more distinct antigens.
  • homologs such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset), or may bind an epitope that is shared between two or more distinct antigens.
  • Vector refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
  • Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems
  • Protein A ligand affinity chromatography refers to an affinity chromatographic method that makes use of the affinity of the IgG binding domains of Protein A ligand for the Fc region of an immunoglobulin molecule. This Fc region comprises human or animal immunoglobulin constant domains CH2 and CH3 or immunoglobulin domains substantially similar to these. Protein A ligand encompasses native protein A from the cell wall of
  • Protein A ligand chromatography involves using Protein A ligand immobilized to a solid support. See Gagnon, Protein A Affinity Chromatography, Purification Tools for Monoclonal Antibodies, pp. 155-198, Validated Biosystems, 1996.
  • the solid support is a non-aqueous matrix onto which Protein A ligand adheres.
  • Such well-known supports include agarose, sepharose, glass, silica, polystyrene, nitrocellulose, charcoal, sand, cellulose and any other suitable material.
  • any suitable well-known method can be used to affix the second protein to the solid support.
  • Such solid supports, with and without immobilized Protein A ligand are readily available from many commercial sources including such as Vector Laboratory (Burlingame, Calif), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif), Amersham Biosciences (part of GE Healthcare, Uppsala, Sweden), Pall (Port Washington, N.Y.) and EMD-Millipore (Billerica, Mass.).
  • Protein A ligand immobilized to a pore glass matrix is commercially available as PROSEP®-A (Millipore).
  • the solid phase may also be an agarose-based matrix.
  • Expression vector refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
  • Polynucleotide refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry.
  • cDNA is a typical example of a synthetic polynucleotide.
  • Polypeptide or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as "peptides”.
  • Variant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.
  • Value refers to the presence of a specified number of binding sites specific for an antigen in a molecule.
  • hexavalent refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.
  • Protein A ligand refers to a naturally occurring or modified Staphylococcal Protein A, and includes engineered Protein A domains.
  • Engineered Protein A may be, for example, Z- domain, variants of Z-domain, Y-domain, or an engineered Protein A that lacks D and E domains.
  • Engineered Protein A domains may be unable to bind (or bind with very low affinity if at all) to the VH3 domain of an immunoglobulin, but can still bind to the CH2-CH3 region of IgGl, IgG2 and IgG4.
  • Z-domain is a synthetic engineered variant of B domain of Staphylococcus aureus protein A having mutations Al V and G29A when compared to the wild-type B domain of protein A.
  • Z-domain comprises the amino acid sequence of SEQ ID NO: 1.
  • Additional Z-domain variants are variants having the amino acid sequences of SEQ ID NOs: 99, 100 and 101, and those described in US2006/0194950.
  • FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU Index as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated. Correspondence between various constant domain numbering systems is available at International ImMunoGeneTics (IMGT) database; Web resources, http://www_imgt_org).
  • IMGT International ImMunoGeneTics
  • compositions of matter multispecific antibodies
  • the invention provides multispecific antibodies and other multimeric CH2-CH3 region containing proteins having asymmetric mutations in the CH2-CH3 region which facilitate their purification using protein A ligand chromatography, polynucleotides encoding them, vectors and host cells, and methods of making and using them.
  • Production and purification of full length bispecific therapeutic antibodies require efficient separation of the bispecific antibodies from excess parental and/or intermediate molecules.
  • Fc mutations have been identified herein which reduce binding of the mutated heavy chain to protein A ligand.
  • Bispecific antibodies having these Fc mutations in asymmetric manner can therefore be purified from the parental antibodies based on their differential elution profile from protein A ligand affinity columns.
  • FcRn is responsible for the transfer of maternal IgG to the fetus and for protecting serum IgG from lysosomal degradation. Both of these processes depend on the ability of FcRn to bind with K D -600 nM to IgG at acidic pH ( ⁇ 6.5) in the recycling endosome and to dissociate at neutral pH, releasing the IgG back into the serum (Roopenian and Akilesh, Nat Rev Immunol 7: 715-725, 2007). IgG binds FcRn at the CH2-CH3 interface, such that a single Fc contains two identical FcRn binding sites.
  • Efforts to modulate protein A ligand binding characteristics of Abs are often associated with significantly decreased serum half -lives since both protein A and the neonatal Fc receptor (FcRn) share a binding site on the Fc.
  • the mutations introduced herein do not reduce binding of the Fc to FcRn and therefore do not reduce serum half -life of the engineered antibodies.
  • One of the introduced mutations, Q311R resulted in slightly enhanced binding to FcRn and increased serum half-life of the antibody.
  • the invention provides for an isolated multispecific antibody comprising a first CH2- CH3 region comprising a mutation Q311R, Q31 IK, T307P/L309Q, T307P/V309Q,
  • the isolated multispecific antibody with asymmetric Q311R, Q31 IK, T307P/L309Q, T307P/V309Q, T307P L309Q/Q311R or T307P/V309Q/Q311R mutations can be efficiently purified from parental antibodies using protein A ligand affinity chromatography.
  • the introduced Q31 IK, T307P/L309Q and T307P/L309Q/Q311R mutations do not reduce binding of the engineered antibodies to FcRn or FcyR, and hence are not expected to alter antibody half-life or effector functions.
  • the introduced Q311R mutation enhanced binding to FcRn and serum half-life of the antibody.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation Q31 IK and a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first
  • CH2-CH3 region comprising a mutation T307P/L309Q and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first
  • CH2-CH3 region comprising a mutation T307P/V309Q and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation T307P/L309Q/Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated multispecific antibody comprising a first
  • the first CH2-CH3 region comprising a mutation T307P/V309Q/Q311R and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the first CH2-CH3 region has reduced binding to protein A ligand when compared to the second CH2-CH3 region.
  • Binding to protein A ligand may be determined experimentally using any suitable method. Such methods may utilize ProteOn XPR36, Biacore 3000 or KinExA instrumentation. The measured affinity may vary if measured under different conditions (e.g., osmolality, pH). Thus, measurements of affinity and other binding parameters (e.g., K D , k on , k 0 ff) are typically made with standardized conditions and a standardized buffer, such as the buffer described herein. Alternatively, binding to protein A ligand may be assessed directly using protein A ligand chromatography using a pH gradient. Molecules with reduced binding to protein A ligand elute at higher pH. An exemplary protein A ligand chromatography may use mAbSelect Sure column (GE Healthcare) and the samples are eluted in 3 steps using buffers containing 50 niM citrate at pH of about pH4.7, pH 4.2 or pH 3.4.
  • protein A ligand comprises Staphylococcal Protein A.
  • protein A ligand comprises Z-domain.
  • Z-domain comprises an amino acid sequence of SEQ ID NO: 1.
  • protein A ligand comprises Y-domain.
  • protein A ligand comprises an amino acid sequence of SEQ ID NO: 1
  • protein A ligand comprises an amino acid sequence of SEQ ID NO: 100.
  • protein A ligand comprises an amino acid sequence of SEQ ID NO: 101.
  • Staphylococcal protein A contains 5 homologous helical IgG-binding domains, denoted E, D, A, B, and C (Uhlen, Guss et al. 1984). Each of these domains is sufficient to bind to the Fc region however spA also binds to the VH region of human VH3 -family members (Romagnani et al, J Immunol 129:596-602, 1982; Sasso et al, J Immunol 147: 1877-1883, 1991). Stability -enhancing mutations introduced into the spA B domain or C domain led to a synthetic Z-domain and Y-domain, respectively, which are resistant to high pH treatment and bind only Fc.
  • Tandem or tetrameric Z-domains, tetrameric Y-domains or native spA have been incorporated into commercial affinity resins such as MabSelect SuRe (GE), TOYOPEARL AF- rProtein A HC-650F and MabSelect Xtra.
  • the multispecific antibody is an IgGl isotype.
  • the multispecific antibody is an IgG2 isotype.
  • the multispecific antibody is an IgG4 isotype.
  • binding of the multispecific antibody to FcyR is comparable to that of the parental antibody without the mutation.
  • FcyR is FcyRI, FcyRIIa, FcyRIIb. and/or FcyRIIIa.
  • FcyR is FcyRI
  • FcyR is FcyRIIa.
  • FcyR is FcyRIIb.
  • FcyR is FcyRIIIa.
  • Exemplary multispecific antibodies with comparable binding to FcyR are multispecific antibodies with Q31 IR or T307P L309Q/Q31 IR mutations.
  • binding of the multispecific antibody to FcRn is comparable to that of the parental antibody without the mutation.
  • Exemplary multispecific antibodies with comparable binding to FcRn are multispecific antibodies with Q31 IK or T307P/L309Q/Q31 IR mutations.
  • binding of the multispecific antibody to FcRn is enhanced when compared to binding of the parental antibody without the mutation to FcRn.
  • Exemplary multispecific antibodies with enhanced binding to FcRn are antibodies with Q311R mutation.
  • the invention also provides for an isolated multispecific antibody comprising a first CH2-CH3 region comprising a mutation Q31 IR, Q31 IK, T307P L309Q, T307P/V309Q, T307P/L309Q/Q31 IR or T307P/V309Q/Q31 IR and a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index, wherein the multispecific antibody further comprises asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are
  • F405W/Y407A and T366W/T394S respectively; L351Y/F405A/Y407V and T394W, respectively;
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are F405L and K409R, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are wild-type and F405L/R409K, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are T366W and T366S/L368A/Y407V, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are T366Y/F405A and T394W/Y407T, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are T366W/F405W and T394S/Y407A, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are F405W/Y407A and T366W/T394S, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are L351Y/F405A/Y407V and T394W, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are T366I/ 392M/T394W and F405A/Y407V, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are T366L/ 392M/T394W and F405A/Y407V, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are L351Y/Y407A and T366A/ 409F, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are L351Y/Y407A and T366V 409F, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are Y407A and T366A/K409F, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are D399K/E356K and K409D/ 392D, respectively.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are D399K/E356K/E357K and K409D/K392D/K370, respectively.
  • Asymmetric stabilizing mutations may be introduced into bispecific or multispecific antibodies to facilitate downstream processes of separating them from excess parental or intermediate molecules.
  • Exemplary asymmetric stabilizing mutations are those that promote Fab arm exchange (e.g., half molecule exchange, exchanging on heavy chain - light chain pair) between two parental antibodies.
  • mutations that favor heterodimer formation of two parental antibody half -molecules either in vitro in cell-free environment or using co-expression are introduced to the heavy chain CH3 interface in each parental antibody.
  • mutations F405L in a first parental antibody and K409R in a second parental antibody may be used to promote Fab arm exchange of IgGl.
  • a wild-type first parental antibody and F405L/R409K mutation in the second parental antibody may be used.
  • knob-in-hole mutations (Genentech) or mutations that introduce electrostatically -matched residues (Chugai, Amgen, NovoNordisk, Oncomed).
  • Exemplary knob-in-hole mutations (expressed as mutated position in the first parental antibody /mutated position in the second parental antibody) are T366Y/F405A,
  • Mutations are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.
  • the multispecific antibody comprises Q311R/F405L mutation in the first CH2-CH3 region and K409R mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises Q311K/F405L mutation in the first CH2-CH3 region and K409R mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/L309Q/F405L mutation in the first CH2-CH3 region and K409R mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises
  • the multispecific antibody comprises Q311R/ 409R mutation in the first CH2-CH3 region and F405L mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises Q31 IK/ K409R mutation in the first CH2-CH3 region and F405L mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/L309Q/ K409R mutation in the first CH2-CH3 region and F405L mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/L309Q/Q311R/ K409R mutation in the first CH2-CH3 region and F405L mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises Q311R mutation in the first
  • the multispecific antibody comprises Q31 IK mutation in the first CH2-CH3 region and F405L/R409K mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/V309Q mutation in the first CH2-CH3 region and F405L/R409K mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/V309Q/Q311R mutation in the first CH2-CH3 region and F405L/R409K mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises Q311R/T366W mutation in the first CH2-CH3 region and T366S/L368A/Y407V mutation in the second CH2-CH3 region. In some embodiments, the multispecific antibody comprises Q311K/T366W mutation in the first CH2-CH3 region and T366S/L368A/Y407V mutation in the second CH2-CH3 region.
  • the multispecific antibody comprises T307P/L309Q/T366W mutation in the first CH2-CH3 region and T366S/L368A/Y407V mutation in the second CH2- CH3 region.
  • the multispecific antibody comprises
  • the multispecific antibody comprises
  • the multispecific antibody comprises
  • the multispecific antibody comprises
  • the multispecific antibody comprises
  • amino acid sequences of exemplary CH2-CH3 regions in the multispecific antibodies of the invention are shown in Table 2 and Table 3.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 2 and 22, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 3 and 22, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 4 and 22, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 5 and 22, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 6 and 23, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 7 and 23, respectively.
  • first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 8 and 23, respectively. In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 9 and 23. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 10 and 24, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 11 and 24, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 12 and 24. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 13 and 24, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 14 and 25, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 15 and 25. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 16 and 25, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 17 and 25, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 18 and 26. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 19 and 26, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 20 and 26, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 21 and 26. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 52 and 54, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 52 and 55, respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 53 and 54. respectively.
  • the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 53 and 55, respectively.
  • first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 56 and 54, respectively. In some embodiments, the first CH2-CH3 region and the second CH2-CH3 region comprise an amino acid sequence of SEQ ID NOs: 56 and 55, respectively.
  • the multispecific antibodies of the invention may further comprise a common light chain to further facilitate downstream processes of separating them from excess parental or intermediate molecules.
  • the multispecific antibody comprises a first light chain and a second light chain.
  • the first light chain and the second light chain have identical amino acid sequences.
  • the multispecific antibody is a bispecific antibody.
  • IgGl CH2-CH3 25 PELLGGPSVFLFPPKPKDTLMISRTPEVTCWV
  • T366S/L368A/Y407 DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
  • the mutations may be transferred to IgG2 and IgG4 isotypes as positions 307, 309 and 311 are conserved across the isotypes except that IgG2 has valine at positon 309. Positions 366, 368 and 407 are also conserved across antibody isotypes. F405L is conserved, however IgG4 has R at position 409. In order to promote Fab arm exchange of human IgG4 antibody, one parental antibody will be engineered to have F405L/R409K mutation and the other parental antibody is wild-type.
  • the multispecific antibody binds at least two antigens.
  • the antigen is ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRLl, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, albumin, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, APOE, AR, AZGP1 (zinc-a-glycoprotein), B7.1, B7.2, BAD, BAFF, BAG1, BAI1, BCL2, BCL6, BDNF, BLNK, BLRl (MDR15), BlyS, BMPl, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, BMPRIA, BMPRIB, BMPR2, BPAGl (plectin), BRCAl, BTLA, C19orfl0 (IL27w), C3, C
  • CD123 CD137, CD164, CD16a, CD16b, CD19, CD1C, CD20, CD200, CD-22, CD24, CD28, CD3, CD30, CD32a, CD32b,CD37, CD38, CD39, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD47, CD52, CD69, CD72, CD73, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD89, CD96, CDHl (E-cadherin), CDHIO, CDH12, CDH13, CDHl 8, CDHl 9, CDH20, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7
  • TDGF1, TEK, TF transferrin receptor
  • TGFA TGFB1, TGFB 111, TGFB2, TGFB3, TGFBI
  • TGFBR1, TGFBR2, TGFBR3, TH1L THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIGIT, TIM-3, TIMP3, tissue factor, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B,
  • the multispecific antibody binds CD3.
  • the multispecific antibody binds CD3 and a tumor antigen.
  • the multispecific antibody binds two antigens wherein the two antigens are any two of PDl, CD27, CD28, NKP46, ICOS, GITR, OX40, CTLA4, LAG3, TIM3, KIRa, CD73, CD39, IDO, BTLA, VISTA, TIGIT, CD96, CD30, HVEM, DNAM-l, LFA, tumor antigen, EGFR, cMet, FGFR, ROR1, CD123, IL1RAP, FGFR, mesothelin, CD3, T cell receptor, CD32b, CD32a, CD 16a, CD 16b, NKG2D, NKP46, CD28, CD47, DLL, CD8, CD89, HLA, B cell receptor or CD 137.
  • the two antigens are any two of PDl, CD27, CD28, NKP46, ICOS, GITR, OX40, CTLA4, LAG3, TIM3, KIRa, CD73, CD39,
  • Additional Fc mutations may be made to the multispecific antibodies of the invention to modulate effector functions and pharmacokinetic properties.
  • effector functions such as antibody -dependent cytotoxicity and phagocytosis
  • immunomodulatory signals such as regulating lymphocyte proliferation and antibody secretion. All of these interactions are initiated through the binding of the Fc region of antibodies or immune complexes to specialized cell surface receptors.
  • FcyRI CD64
  • FcyRIIA CD32A
  • FcyRIII CD16
  • FcyRIIB CD32B
  • Binding to the FcRn receptor modulates antibody half-life.
  • the multispecific antibody of the invention further comprises at least one mutation that modulates binding of the antibody to FcyR. In some embodiments, the multispecific antibody of the invention further comprises at least one mutation that modulates binding of the antibody to or FcRn.
  • Exemplary mutations that increase half-life of the multispecific antibody are mutations M428L/N434S, M252Y/S254T/T256E, T250Q/M428L, N434A and T307A/E380A/N434A.
  • Exemplary mutations that reduce half -life of the multispecific antibody are mutations H435A, P257I/N434H, D376V/N434H, M252Y/S254T/T256E/H433K/N434F, T308P/N434A and H435R.
  • the multispecific antibody of the invention comprises at least one mutation that reduces binding of the antibody to an activating Fey receptor (FcyR) and/or reduces Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), antibody- dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP).
  • FcyR activating Fey receptor
  • CDC complement dependent cytotoxicity
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • ADCP phagocytosis
  • Exemplary mutations that reduce binding of the multispecific antibody of the invention to activating FcyR and/or minimize antibody effector functions are L234A/L235A on IgGl, V234A,/G237A/ P238S/H268A/V309L/A330S/P33 IS on IgG2, F234A/L235A on IgG4, S228P/F234A/ L235A on IgG4, N297A on all Ig isotypes, V234A/G237A on IgG2,
  • H268Q/V309L/ A330S/P33 IS on IgG2, S267E L328F on IgGl, L234F L235E D265A on IgGl, L234A/L235A/G237A/P238S/H268A/A330S/P331S on IgGl,
  • Exemplary mutations that increase binding of the multispecific antibody of the invention to an activating Fey and/or enhance antibody effector functions are S239D/I332E,
  • Well-known S228P may be made in IgG4 antibodies to enhance IgG4 stability.
  • Antibody -dependent cellular cytotoxicity is a mechanism for inducing cell death that depends upon the interaction of antibody -coated target cells with effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
  • effector cells possessing lytic activity such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (FcyR) expressed on effector cells.
  • FcyR Fc gamma receptors
  • NK cells express FcyRIIIa
  • monocytes express FcyRI, FcyRII and FcyRIIIa.
  • Death of the antibody -coated target cells occurs as a result of effector cell activity through the secretion of membrane pore-forming proteins and proteases.
  • the antibodies may be added to cells expressing the desired antigen in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis may be detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins
  • exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • target cells include cells expressing the desired antigen either endogenously or recombinantly. In an exemplary assay, target cells are used with a ratio of 1 target cell to 50 effector cells.
  • Target cells are pre-labeled with BATDA (PerkinElmer) for 20 minutes at 37°C, washed twice and resuspended in DMEM, 10% heat-inactivated FBS, 2mM L-glutamine (all from Invitrogen).
  • Target (lxlO 4 cells) and effector cells (0.5xl0 6 cells) are combined and ⁇ of cells are added to the wells of 96-well U- bottom plates. An additional 100 ⁇ is added with or without the test antibodies. The plates are centrifuged at 200g for 3 minutes, incubated at 37°C for 2 hours, and then centrifuged again at 200g for 3 minutes.
  • ADCP Antibody -dependent cellular phagocytosis
  • phagocytic cells such as macrophages or dendritic cells.
  • ADCP may be evaluated by using monocyte-derived macrophages as effector cells and Daudi cells (ATCC ® CCL-213TM) or B cell leukemia or lymphoma or tumor cells expressing the desired antigen as target cells engineered to express GFP or other labeled molecule.
  • Effectontarget cell ratio may be for example 4: 1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the invention. After incubation, cells may be detached using accutase.
  • Macrophages may be identified with anti-CD 1 lb and anti-CD 14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD 11 + CD 14 + macrophages using standard methods.
  • “Complement-dependent cytotoxicity” refers to a mechanism for inducing cell death in which the Fc effector domain of a target-bound antibody binds and activates complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
  • complement receptors e.g., CR3
  • CDC may be measured for example by plating Daudi cells at 1 ⁇ 10 5 cells/well (50 ⁇ /well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 ⁇ of test antibodies to the wells at final concentration between 0-100 ⁇ g/ml, incubating the reaction for 15 min at room temperature, adding 11 ⁇ of pooled human serum to the wells, and incubation the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.
  • Additional mutations may further be made to the multispecific antibodies of the invention that enhance binding of the antibody to FcyRIIb.
  • Exemplary such mutations are mutations S267E, S267D, S267E/I332E, S267E/L328F, G236D/S267E and
  • mutations enhancing binding to activating FcyR and reducing binding to inhibitory FcyRIIb may be engineered into antibodies to be used to enhance immune responses in a subject, such as for the treatment of cancers and infections.
  • Mutations reducing binding to activating FcyR or enhancing binding to the inhibitory FcyRIIb may be engineered into antibodies which are used to dampen immune responses in a subject, such as for the treatment of inflammatory or autoimmune disease.
  • Mutations enhancing binding to inhibitory FcyRIIb may also be introduced into agonistic antibodies that bind TNFR superfamily members to enhance their agonistic activity.
  • the ability of the multispecific antibodies of the invention to induce ADCC may be enhanced by engineering their oligosaccharide component.
  • Human IgGl is N-glycosylated at Asn297 with the majority of the glycans in the well-known biantennary GO, GOF, Gl, GIF, G2 or G2F forms.
  • Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. The removal of the core fucose from the biantennary complex- type oligosaccharides attached to the Fc regions enhances the ADCC of antibodies via improved FcyRIIIa binding without altering antigen binding or CDC activity.
  • Such mAbs may be achieved using different methods reported to lead to the successful expression of relatively high defucosylated antibodies bearing the biantennary complex-type of Fc oligosaccharides such as control of culture osmolality, application of a variant CHO line Lecl3 as the host cell line, application of a variant CHO line EB66 as the host cell line, application of a rat hybridoma cell line YB2/0 as the host cell line, introduction of small interfering RNA specifically against the a 1,6-fucosyltrasferase ( FUT8) gene, or coexpression of P-l,4- -acetylglucosaminyltransferase III and Golgi a-mannosidase II or a potent alpha-mannosidase I inhibitor, kifunensine.
  • FUT8 1,6-fucosyltrasferase
  • the multispecific antibodies of the invention have a biantennary glycan structure with fucose content of about between 0% to about 15%, for example 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
  • the multispecific antibodies of the invention have a biantennary glycan structure with fucose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0%.
  • “Fucose content” means the amount of the fucose monosaccharide within the sugar chain at Asn297. The relative amount of fucose is the percentage of fucose-containing structures related to all glycostructures.
  • N-glycosidase F treated sample e.g. complex, hybrid and oligo- and high-mannose structures
  • MALDI-TOF MALDI-TOF of N-glycosidase F treated sample
  • UPLC HPLC
  • UPLC-MS fluorescence detection and/or HPLC-MS
  • digestion of the mAb to constituent peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), and subsequent separation, detection and quantitation by HPLC-MS (UPLC-MS) or 5) separation of the mAb
  • the oligosaccharides released may be labeled with a fluorophore, separated and identified by various complementary techniques which allow fine characterization of the glycan structures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry by comparison of the experimental masses with the theoretical masses, determination of the degree of sialylation by ion exchange HPLC (GlycoSep C), separation and quantification of the oligosaccharide forms according to hydrophilicity criteria by normal-phase HPLC (GlycoSep N), and separation and quantification of the oligosaccharides by high performance capillary electrophoresis-laser induced fluorescence (HPCE-LIF).
  • MALDI matrix-assisted laser desorption ionization
  • Low fucose or “low fucose content” refers to antibodies with fucose content of about 0% - 15%.
  • Normal fucose or 'normal fucose content refers to antibodies with fucose content of about over 50%, typically about over 60%, 70%, 80% or over 85%.
  • the multispecific antibodies of the invention may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro.
  • the antibodies of the invention described herein may be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation may be carried out by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function.
  • Multispecific antibodies of the invention may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the invention.
  • Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein/protein interface interactions that have impact upon the HC and LC pairing, (3) burial of polar and charged residues, (4) H-bonding network for polar and charged residues; and (5) surface charge and polar residue distribution among other intra- and inter-molecular forces (Worn and Pluckthun 2001).
  • Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modeling in certain cases, and the effect of the residues on antibody stability may be tested by generating and evaluating variants harboring mutations in the identified residues.
  • One of the ways to increase antibody stability is to raise the thermal transition midpoint (T m ) as measured by differential scanning calorimetry (DSC).
  • T m thermal transition midpoint
  • DSC differential scanning calorimetry
  • the protein T m is correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold.
  • Formulation studies suggest that a Fab T m has implication for long-term physical stability of a corresponding mAb.
  • CTL C-terminal lysine
  • CTL removal may be controlled to less than the maximum level by control of concentration of extracellular Zn 2+ , EDTA or EDTA - Fe 3+ as described in U.S. Patent Publ. No. US20140273092.
  • CTL content in antibodies can be measured using known methods.
  • the multispecific antibodies of the invention have a C-terminal lysine content of about 10% to about 90%, about 20% to about 80%, about 40% to about 70%, about 55% to about 70%, or about 60%.
  • the multispecific antibodies of the invention have a C-terminal lysine content of about 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation Q311R, Q31 IK, T307P/L309Q, T307P/V309Q, T307P/L309Q/Q311R or T307P/V309Q/Q311R, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation Q311R, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation Q31 IK, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation T307P L309Q, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation T307P/V309Q, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation T307P/L309Q/Q311R, wherein residue numbering is according to the EU Index.
  • the invention also provides for an isolated antibody comprising two heavy chains having identical amino acid sequences and two light chains, wherein the two identical heavy chains comprises a mutation T307P/V309Q/Q311R, wherein residue numbering is according to the EU Index.
  • the isolated antibody is useful as a parental antibody for generating the multispecific antibodies of the invention.
  • the isolated antibody further comprises a mutation F405L, K409R, F405L/R409K, T366W or T366S/L368A/Y407V.
  • the isolated antibody is an IgGl, an IgG2 or an IgG4 isotype.
  • the engineered multispecific antibodies of the invention that have altered amino acid sequences when compared to the parental multispecific antibodies may be generated using standard cloning and expression technologies. For example, site-directed mutagenesis or PCR- mediated mutagenesis may be performed to introduce the mutation(s) and the effect on antibody binding or other property of interest, may be evaluated using well known methods and the methods described herein in the Examples.
  • Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al, (2003) N EnglJ Med 348:602-08).
  • the extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al, (2011) Genes and
  • Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody.
  • Table 4 shows select IgGl, IgG2 and IgG4 allotypes.
  • the multispecific antibodies of the invention are of G2m(n), G2m(n-), G2m(n)/(n-), nG4m(a), Glm(17) or Glm(17,l) allotype. Table 4.
  • the multispecific antibodies of the invention may be generated using standard molecular biology techniques and promoting Fab arm exchange of the parental antibodies.
  • the multispecific antibodies of the invention may be purified using protein A ligand affinity chromatography.
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain or fragment thereof comprising a mutation Q31 IR, Q31 IK,
  • a first parental antibody comprising the first heavy chain or fragment thereof comprising the mutation Q31 IR, Q31 IK, T307P L309Q, T307P/V309Q, T307P/L309Q/Q31 IR or T307P/V309Q/Q31 IR and a first light chain;
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain or fragment thereof comprising a mutation T307P/L309Q and a second heavy chain or fragment thereof comprising wild-type amino acid residue at positions 307 and 309, comprising
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain or fragment thereof comprising a mutation T307P/V309Q and a second heavy chain or fragment thereof comprising wild-type amino acid residue at positions 307 and 309, comprising
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain or fragment thereof comprising a mutation T307P/L309Q/Q311R and a second heavy chain or fragment thereof comprising wild-type amino acid residue at positions 307, 309 and 311, comprising
  • the invention also provides for a method of making an isolated multispecific antibody comprising a first heavy chain or fragment thereof comprising a mutation T307P/V309Q/Q311R and a second heavy chain or fragment thereof comprising wild-type amino acid residue at positions 307, 309 and 311, comprising
  • the VH and the VL regions of the multispecific antibodies may be derived from existing VH/VL regions of antibodies specific to a desired antigen, or from VH/VL domains of parental antibodies generated de novo.
  • the parental antibodies may be generated de novo using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975 may be used to generate them.
  • a mouse or other host animal such as a hamster, rat or monkey, is immunized with an antigen followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding,
  • Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, and affinity for the antigen.
  • Transgenic mice carrying human immunoglobulin (Ig) loci in their genome may be used to generate the parental antibodies against a desired antigen, and are described in for example Int. Pat. Publ. No. WO90/04036, U.S. Pat. No. 6150584, Int. Pat. Publ. No. W099/45962, Int. Pat. Publ. No. WO02/066630, Int. Pat. Publ. No. WO02/43478, Lonberg et al, Nature 368:856-9, 1994; Green et al, Nature Genet 7: 13-21, 1994; Green & Jakobovits, Exp. Med.
  • the endogenous immunoglobulin loci in such mice may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the mouse genome using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron
  • the parental antibodies may also be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions.
  • the parental antibodies may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al, JMol Biol 397:385-96, 2010 and Int. Pat. Publ. No. WO09/085462).
  • the libraries may be screened for phage binding to the desired antigen and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs.
  • phage display methods for isolating human antibodies are described in for example: U.S. Patent Nos. 5,223,409; 5,403,484, 5,571,698, 5,427,908, 5, 580,717, 5,969,108, 6,172,197, 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.
  • the isolated VH/VL regions may be cloned as any Ig isotype or a portion of antibody constant domain, such as a CH2-CH3 region using standard cloning methods. Fc mutations may be introduced to the parental antibodies using standard methods.
  • the first parental antibody and the second parental antibody are provided as purified antibodies.
  • the first parental antibody and the second parental antibody are provided in a cell culture medium collected from cells expressing the first parental antibody and the second parental antibody.
  • the first parental antibody and the second parental antibody are co-expressed in a cell.
  • an incubation step is performed.
  • incubation is performed at a temperature of about 20°C to about
  • incubation is performed at a temperature of about 25°C to about
  • incubation is performed at a temperature of about 25°C to about 37°C about ninety minutes to about six hours.
  • a reducing agent is added during the incubation step.
  • the reducing agent is 2-mercaptoethylamine (2-MEA).
  • the reducing agent is dithiothreitol (DTT).
  • the reducing agent is dithioerythritol (DTE).
  • the reducing agent is glutathione.
  • the reducing agent is tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the reducing agent is L-cysteine.
  • the reducing agent is beta-mercaptoethanol.
  • the reducing agent is present at a concentration of about 10 mM to about 100 mM.
  • 2-MEA is present at a concentration of about 10 mM to about 100 mM.
  • 2-MEA is present at a concentration of about 25 mM to about 75 mM.
  • incubation for at least 90 min at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.
  • protein A ligand chromatography employs a pH gradient.
  • the pH gradient is from about pH 7.0 to about pH 3.0.
  • the pH gradient is from about pH 4.6 to about pH 3.4.
  • the multimeric antibody elutes between about pH 4.4 to about pH
  • the pH gradient is a step gradient of pH 4.6, pH 4.1 and pH 3.4.
  • protein A ligand chromatography employs a citrate buffer. In some embodiments, protein A ligand chromatography employs a 50 mM citrate buffer.
  • protein A ligand chromatography employs an acetate buffer. In some embodiments, protein A ligand chromatography employs a 40 mM acetate buffer.
  • Protein A chromatography may be carried out using mAb Select Sure columns (GE Healthcare) or in batch mode. Culture supernatants are loaded onto the column directly without additional processing, according to the manufacturer's column specifications. Antibodies are eluted using pH step gradient using buffers containing 50 mM citrate pH 4.7, pH 4.2 or pH 3.4. Elution fractions are collected and concentrated to > 1 mg/mL prior to analysis. Purity of the isolated multimeric antibody can be assessed using hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • compositions of matter multimeric proteins of the invention
  • the mutations identified herein may be used to isolate any multimeric protein from its parental proteins as long as the multimeric protein has at least two polypeptide chains each having a CH2-CH3 region with asymmetrical Q311R, Q31 IK, T307P/L309Q, T307P/V309Q, T307P/L309Q/Q311R or T307P/V309Q/Q311R mutations.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation Q311R, Q31 IK, T307P/L309Q, T307P/V309Q, T307P/L309Q/Q311R or
  • T307P/V309Q/Q311R and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation Q311R and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation Q31 IK and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at position 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation T307P/L309Q and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation T307P/V309Q and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307 and 309, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation T307P/L309Q/Q311R and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the invention also provides for a multimeric protein comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first CH2-CH3 region comprising a mutation T307P/V309Q/Q311R and the second polypeptide comprises a second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311, wherein residue numbering is according to the EU Index.
  • the first CH2-CH3 region and the second CH2-CH3 region are an IgGl isotype.
  • the first CH2-CH3 region and the second CH2-CH3 region are an IgG2 isotype.
  • the first CH2-CH3 region and the second CH2-CH3 region are an IgG4 isotype.
  • the first CH2-CH3 region has reduced binding to protein A ligand when compared to the second CH2-CH3 region.
  • protein A ligand comprises Staphylococcal Protein A.
  • protein A ligand comprises Z-domain.
  • protein A ligand comprises Y-domain.
  • Z-domain comprises an amino acid sequence of SEQ ID NO: 1.
  • protein A ligand comprises an amino acid sequence of SEQ ID Nos: 99, 100 or 101.
  • the multimeric protein further comprises asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region.
  • the asymmetric stabilizing mutations in the first CH2-CH3 region and in the second CH2-CH3 region or in the second CH2-CH3 region and in the first CH2-CH3 region are
  • T366L/ 392M/T394W and F405A/Y407V respectively; L351Y/Y407A and T366A/K409F, respectively;
  • the first CH2-CH3 region and the second CH2-CH3 comprise an amino acid sequence of
  • the first CH2-CH3 region and/or the second CH2-CH3 region is coupled to a heterologous protein.
  • the heterologous protein is a peptide. In some embodiments, the heterologous protein is an extracellular domain of a receptor. In some embodiments, the heterologous protein is an extracellular domain of a ligand. In some embodiments, the heterologous protein is a secreted protein.
  • the heterologous protein is a scFv.
  • the heterologous protein is a heavy chain variable region (VH). In some embodiments, the heterologous protein is a light chain variable region (VL). In some embodiments, the heterologous protein is a fibronectin type III domain.
  • the heterologous protein is a fynomer.
  • the heterologous protein is coupled to the N-terminus of the first CH2-CH3 region and/or the second CH2-CH3 region, optionally via a linker.
  • the heterologous protein is coupled to the C-terminus of the first CH2-CH3 region and/or the second CH2-CH3 region, optionally via a linker.
  • the linker comprises an amino acid sequence of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 92, 93, 94, 95, 96, 97 or 98.
  • the multimeric protein is an antibody.
  • the antibody is multispecific.
  • the antibody is bispecific.
  • the antibody is monospecific.
  • the multimeric protein contains two polypeptide chains.
  • the multimeric protein contains three polypeptide chains.
  • the multimeric protein contains four polypeptide chains.
  • peptide (P) may be an extracellular domain of a receptor, an extracellular domain of a ligand, a secreted protein, a scFv, a Fab, a heavy chain variable region (VH), a light chain variable region (VL), a fibronectin type III domain or a fynomer.
  • linker (L) may optionally be absent.
  • Exemplary linkers are shown in Table 6.
  • Asterix (*) in the table indicates that the two CH2-CH3 domains harbor asymmetrical mutations as have been described herein.
  • the multimeric proteins of the invention may be further modified as described herein for multispecific antibodies using standard methods.
  • the multimeric proteins of the invention may be made using standard cloning methods.
  • the invention also provides for an isolated polynucleotide encoding any of the CH2- CH3 regions, antibody heavy chains, antibody light chains or polypeptides of the multimeric proteins of the invention.
  • the invention also provides for an isolated polynucleotide
  • T307P/V309Q/Q311R and the second CH2-CH3 region comprising a wild-type amino acid residue at positions 307, 309 and 311;
  • polynucleotide sequences of the invention may be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell.
  • the polynucleotide may be a cDNA.
  • the invention also provides for a vector comprising the polynucleotide of the invention.
  • vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the synthetic polynucleotide of the invention into a given organism or genetic background by any means.
  • the polynucleotides of the invention may be operably linked to control sequences in the expression vector(s) that ensure the expression of the CH2-CH3 regions the polynucleotides encode.
  • control sequences include signal sequences, promoters (e.g. naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the antibody.
  • the vector comprises the polynucleotides of SEQ ID Nos: 27, and 47.
  • the vector comprises the polynucleotides of SEQ ID Nos: 28 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 29 and 47.
  • the vector comprises the polynucleotides of SEQ ID NOs: 30 and 47. In some embodiments, the vector comprises the polynucleotides of SEQ ID NOs: 31 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 32 and
  • the vector comprises the polynucleotides of SEQ ID NOs: 33 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 34 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 35 and 49.
  • the vector comprises the polynucleotides of SEQ ID Nos: 36 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 37 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 38 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 39 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 40 and 50.
  • the vector comprises the polynucleotides of SEQ ID Nos: 41 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 42 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 43 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 44 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 45 and 51.
  • the vector comprises the polynucleotides of SEQ ID NOs: 46 and
  • the vector comprises the polynucleotides of SEQ ID NOs: 87 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 87 and
  • the vector comprises the polynucleotides of SEQ ID NOs: 88 and
  • the vector comprises the polynucleotides of SEQ ID NOs: 88 and
  • the vector comprises the polynucleotides of SEQ ID NOs: 92 and
  • the vector comprises the polynucleotides of SEQ ID Nos: 92 and
  • Table 7 shows the cDNA sequences of exemplary CH2-CH3 regions. Table 7.
  • IgGl CH2- 29 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 31 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 32 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 33 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 35 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 36 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 37 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 38 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 39 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA Q311K/ CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC T366W ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
  • IgGl CH2- 40 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 41 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 42 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 43 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 44 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • IgGl CH2- 45 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
  • T307P L30 CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC 9Q/ ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGG
  • T366S L36 ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC 8A/Y407V GGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA
  • IgGl CH2- 46 CCTGAACTGCTGGGGGGACCGTCAGTCTTCCTCTTCC CH3 CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA

Abstract

La présente invention concerne des anticorps multispécifiques modifiés et d'autres protéines multimères avec des mutations de région CH2-CH3 asymétriques et des procédés de fabrication et d'utilisation de ceux-ci.
PCT/IB2018/053997 2017-06-05 2018-06-04 Anticorps multispécifiques modifiés et autres protéines multimères avec des mutations de région ch2-ch3 asymétriques WO2018224951A2 (fr)

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AU2018281045A AU2018281045A1 (en) 2017-06-05 2018-06-04 Engineered multispecific antibodies and other multimeric proteins with asymmetrical CH2-CH3 region mutations
KR1020197038922A KR20200014379A (ko) 2017-06-05 2018-06-04 비대칭 ch2-ch3 영역 돌연변이를 갖는 조작된 다중특이성 항체 및 다른 다량체 단백질
JP2019566965A JP2020522266A (ja) 2017-06-05 2018-06-04 非対称なｃｈ２−ｃｈ３領域の変異を有する、操作された多重特異性抗体及び他の多量体タンパク質
BR112019025583-4A BR112019025583A2 (pt) 2017-06-05 2018-06-04 Anticorpos multiespecíficos geneticamente modificados e outras proteínas multiméricas com mutações assimétricas na região ch2-ch3
CN201880037442.XA CN110785185A (zh) 2017-06-05 2018-06-04 具有非对称ch2-ch3区突变的工程化多特异性抗体和其他多聚体蛋白
EP18814314.3A EP3634486A4 (fr) 2017-06-05 2018-06-04 Anticorps multispécifiques modifiés et autres protéines multimères avec des mutations de région ch2-ch3 asymétriques
CA3065171A CA3065171A1 (fr) 2017-06-05 2018-06-04 Anticorps multispecifiques modifies et autres proteines multimeres avec des mutations de region ch2-ch3 asymetriques
RU2019144115A RU2804031C2 (ru) 2017-06-05 2018-06-04 Сконструированные мультиспецифические антитела и другие мультимерные белки с асимметричными мутациями в области ch2-ch3
MX2019014576A MX2019014576A (es) 2017-06-05 2018-06-04 Anticuerpos multiespecíficos manipulados genéticamente y otras proteínas multiméricas con mutaciones asimétricas en la región ch2-ch3.

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